猪羊水干细胞生物学特性检测及其特异性诱导分化研究
摘要
羊水来源干细胞(amniotic fluid stem cells,AFScs)是一种存在于胎儿羊水中Oct4阳性的多能性干细胞。该细胞具有增殖能力强、分化潜能高和免疫原性低等优点,在生物学和医学研究领域具有广泛的应用前景。本实验从不同怀孕期长白二元杂交母猪的羊水标本中分离猪羊水干细胞,探讨了猪胎龄对细胞贴壁率和细胞活性的影响,并筛选优化了猪AFS细胞的培养体系。同时,通过RT-PCR、流式细胞仪、免疫荧光及体内外诱导分化等技术对猪AFS细胞进行生物学特性检测,并在体外特异性诱导猪AFS细胞向心肌细胞分化。上述研究为大动物羊水干细胞的基础研究和临床应用提供了可借鉴的材料。
一、猪AFS细胞培养方法的建立
收集长白二元杂交怀孕母猪的羊水标本,离心收集细胞,加入猪AFS细胞培养液培养。对血清浓度及基础培养液进行筛选,以期获得最佳的培养体系,并且探讨了胎龄对细胞贴壁率和细胞活性的影响。
结果表明:基础培养液α-MEM+10%胎牛血清为猪AFS细胞的最佳培养体系;胎龄对细胞贴壁率和细胞活性均有明显影响,综合分析,从胎龄为40-70d的猪胎儿羊水中分离出的羊水干细胞贴壁率高,增殖能力强且细胞活性好。
二、猪AFS细胞的生物学特性研究
对分离培养的猪AFS细胞进行生长曲线和细胞周期检测,并通过RT-PCR、免疫荧光、流式细胞仪等技术对其生物学特性进行研究。
结果表明:猪羊水干细胞在体外培养过程中,3天后达到对数增长期,6天后细胞增殖达到顶峰,表现出了极强的增殖能力;流式细胞周期检测显示处于G1期和S期的细胞比例较高,且在体外多次传代后仍保持正常的二倍体核型;猪AFS细胞表达胚胎干细胞标志基因Oct4、Nanog、SSEA-4、Tra-60、Tra-81、CD117和主要组织相容性抗原HLA-ABC,不表达SSEA-1,表达间充质特异标志基因CD44、CD90、CD166,不表达造血干细胞标志基因CD45、CD34。
三、猪AFS细胞体内外诱导分化研究
在体外将猪AFS细胞形成类胚体,并通过AP染色和RT-PCR对形成的类胚体进行检测;特异性诱导猪AFS细胞向脂肪细胞、神经细胞、精原细胞和心肌细胞分化,以检测其体外分化潜能;通过裸鼠注射实验检测猪AFS细胞的体内分化潜能;
结果表明:猪AFS细胞在体外能形成类胚体,AP染色阳性,且表达三胚层特异标志基因;在体外猪AFS细胞可诱导为脂肪细胞、神经细胞、精原细胞和心肌细胞,经检测有目的细胞基因的表达;将猪AFS细胞注射到裸鼠皮下6-8周后,注射部位未发现肿瘤结节的形成,说明猪AFS细胞在体内无致瘤性。
Amniotic Fluid Stem(AFS)cells a pluripotent stem cells which exist in amniotic fluid and express Oct4. These cells show many charming features, such as robust proliferation potention, pluripotent ability and low immunogenicity, and have wildly promise in the fields of biology and medicine. This study isolated the porcine AFS cells from amniotic fluid in different development stage embryos of landrace crossbred, and investigated the relationship of age of embryos to the adhere-rate and activity of porcine AFS cells, and further optimized the cultrue condition of porcine AFS cells. Further more, characterization of porcine AFS cells using PT-PCR, Flow-cytometry, immunofluorescence and differentiation in vivo and vitro,. These results has provided useful experience for the basic research and application of amniotic fluid stem cells of large animals.
1. Establishment the culture system to isolation of porcine AFS cells
Collected porcine AFS cells through centrifugation from amniotic fluid which derived from fetus,and cultured these cells in specific AFS cells medium. Using several kinds of basic medium and different concentration of FBS, the optimized culture system was selected. Besides, probed the effect of fetus age to the adhere-rate of cells and cell activity. The result suggested thatα-MEM plus 10% FBS is the best culture condition for porcine AFS cells, and the effect of fetus age to the adhere-rate of cells and cell active is visible. The AFS cells derived from amniotic fluid of 40d-70d fetus prefer to adhere, and also has great proliferated potency.
2. Characterization of porcine AFS cells
The growth curve and cell cycle of porcine AFS cells was detected, and characterized the biological features of which using PT-PCR, immunofluorescence and flow cytometry porcine. The result showed that porcine AFS cells displayed robust proliferated potential in vitro culture, and 3 days later cell accessed to exponential growth, after 6 days, the number of cells reached culmination. The result of flow cytometry revealed that porcine AFS cells had normal karyocyte and high proportion of which stayed in the G1 and S stage. Furthermore, porcine AFS cells expressed many marker genes of ES cells, such as Oct4、Nanog、SSEA-4、Tra-60、Tra-81 and CD117 plus crucial MHC antigen HLA-ABC, but not for SSEA1. Besides these cells also expressed mesenchymal stem cells markers CD44, CD90 and CD166, and none of hematopoietic stem cells markers CD45 and CD 34.
3. Induction of porcine AFS cells to differentiate
Formed embryoid bodies (EBs) through suspended culture in vitro, and followed detection of EBs using AP staining and RT-PCR, and induced to differentiate into adipocytes、neurocytes、premeiotic germ-like cells and myocardial cells. Furthermore, porcine AFS cells were injected into nude mouse and detected the differentiated capability of which in vivo. The results diplayed that porcine AFS cells had the ability of Ebs formation in vitro and the EBs were positive for AP staining and expressed many markers of three germ layers. Furthermore, porcine AFS cells could be induced to differentiate into adipocytes、neurocytes、premeiotic germ-like cells and myocardial cells in vitro and expressed specific genes in target cells. After injected porcine AFS cells into subcutaneous of nude mouse, 6-8 weeks later, none of teratoma was appeared in the body, which suggested that porcine AFS cells have not the risk of tumorigenicity.
一、猪AFS细胞培养方法的建立
收集长白二元杂交怀孕母猪的羊水标本,离心收集细胞,加入猪AFS细胞培养液培养。对血清浓度及基础培养液进行筛选,以期获得最佳的培养体系,并且探讨了胎龄对细胞贴壁率和细胞活性的影响。
结果表明:基础培养液α-MEM+10%胎牛血清为猪AFS细胞的最佳培养体系;胎龄对细胞贴壁率和细胞活性均有明显影响,综合分析,从胎龄为40-70d的猪胎儿羊水中分离出的羊水干细胞贴壁率高,增殖能力强且细胞活性好。
二、猪AFS细胞的生物学特性研究
对分离培养的猪AFS细胞进行生长曲线和细胞周期检测,并通过RT-PCR、免疫荧光、流式细胞仪等技术对其生物学特性进行研究。
结果表明:猪羊水干细胞在体外培养过程中,3天后达到对数增长期,6天后细胞增殖达到顶峰,表现出了极强的增殖能力;流式细胞周期检测显示处于G1期和S期的细胞比例较高,且在体外多次传代后仍保持正常的二倍体核型;猪AFS细胞表达胚胎干细胞标志基因Oct4、Nanog、SSEA-4、Tra-60、Tra-81、CD117和主要组织相容性抗原HLA-ABC,不表达SSEA-1,表达间充质特异标志基因CD44、CD90、CD166,不表达造血干细胞标志基因CD45、CD34。
三、猪AFS细胞体内外诱导分化研究
在体外将猪AFS细胞形成类胚体,并通过AP染色和RT-PCR对形成的类胚体进行检测;特异性诱导猪AFS细胞向脂肪细胞、神经细胞、精原细胞和心肌细胞分化,以检测其体外分化潜能;通过裸鼠注射实验检测猪AFS细胞的体内分化潜能;
结果表明:猪AFS细胞在体外能形成类胚体,AP染色阳性,且表达三胚层特异标志基因;在体外猪AFS细胞可诱导为脂肪细胞、神经细胞、精原细胞和心肌细胞,经检测有目的细胞基因的表达;将猪AFS细胞注射到裸鼠皮下6-8周后,注射部位未发现肿瘤结节的形成,说明猪AFS细胞在体内无致瘤性。
Amniotic Fluid Stem(AFS)cells a pluripotent stem cells which exist in amniotic fluid and express Oct4. These cells show many charming features, such as robust proliferation potention, pluripotent ability and low immunogenicity, and have wildly promise in the fields of biology and medicine. This study isolated the porcine AFS cells from amniotic fluid in different development stage embryos of landrace crossbred, and investigated the relationship of age of embryos to the adhere-rate and activity of porcine AFS cells, and further optimized the cultrue condition of porcine AFS cells. Further more, characterization of porcine AFS cells using PT-PCR, Flow-cytometry, immunofluorescence and differentiation in vivo and vitro,. These results has provided useful experience for the basic research and application of amniotic fluid stem cells of large animals.
1. Establishment the culture system to isolation of porcine AFS cells
Collected porcine AFS cells through centrifugation from amniotic fluid which derived from fetus,and cultured these cells in specific AFS cells medium. Using several kinds of basic medium and different concentration of FBS, the optimized culture system was selected. Besides, probed the effect of fetus age to the adhere-rate of cells and cell activity. The result suggested thatα-MEM plus 10% FBS is the best culture condition for porcine AFS cells, and the effect of fetus age to the adhere-rate of cells and cell active is visible. The AFS cells derived from amniotic fluid of 40d-70d fetus prefer to adhere, and also has great proliferated potency.
2. Characterization of porcine AFS cells
The growth curve and cell cycle of porcine AFS cells was detected, and characterized the biological features of which using PT-PCR, immunofluorescence and flow cytometry porcine. The result showed that porcine AFS cells displayed robust proliferated potential in vitro culture, and 3 days later cell accessed to exponential growth, after 6 days, the number of cells reached culmination. The result of flow cytometry revealed that porcine AFS cells had normal karyocyte and high proportion of which stayed in the G1 and S stage. Furthermore, porcine AFS cells expressed many marker genes of ES cells, such as Oct4、Nanog、SSEA-4、Tra-60、Tra-81 and CD117 plus crucial MHC antigen HLA-ABC, but not for SSEA1. Besides these cells also expressed mesenchymal stem cells markers CD44, CD90 and CD166, and none of hematopoietic stem cells markers CD45 and CD 34.
3. Induction of porcine AFS cells to differentiate
Formed embryoid bodies (EBs) through suspended culture in vitro, and followed detection of EBs using AP staining and RT-PCR, and induced to differentiate into adipocytes、neurocytes、premeiotic germ-like cells and myocardial cells. Furthermore, porcine AFS cells were injected into nude mouse and detected the differentiated capability of which in vivo. The results diplayed that porcine AFS cells had the ability of Ebs formation in vitro and the EBs were positive for AP staining and expressed many markers of three germ layers. Furthermore, porcine AFS cells could be induced to differentiate into adipocytes、neurocytes、premeiotic germ-like cells and myocardial cells in vitro and expressed specific genes in target cells. After injected porcine AFS cells into subcutaneous of nude mouse, 6-8 weeks later, none of teratoma was appeared in the body, which suggested that porcine AFS cells have not the risk of tumorigenicity.
引文
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